The blood transcriptome of childhood malaria.

BACKGROUND: Transcriptomic research of blood cell lineages supports the understanding of distinct features of the immunopathology in human malaria. METHODS: We used microarray hybridization, validated by real-time RT-PCR to analyze whole blood gene expression in healthy Gabonese children and children with various conditions of Plasmodium falciparum infection, including i) asymptomatic infection, ii) uncomplicated malaria, iii) malaria associated with severe anemia and iv) cerebral malaria. FINDINGS: Our data indicate that the expression profile of 22 genes significantly differed among the investigated groups. Immunoglobulin production, complement regulation and IFN beta signaling, in particular IRF7 and ISRE binding signatures in the corresponding genes, were most conspicuous. Down-regulation in cerebral malaria seems to rely on AhRF, GABP and HIF1 hypoxia transcription factors. ARG1, BPI, CD163, IFI27, HP and TNFAIP6 transcript levels correlated positively with lactatemia, and negatively with hemoglobin concentrations. INTERPRETATION: Differences in gene expression profile reflect distinct immunopathological mechanisms of P. falciparum infection. They emerge as potential prognostic markers for early therapeutic measures and need to be validated further. FUND: This work was supported by a grant of the NGFN (Nationales Genomforschungsnetz 01GS0114) and by a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) PhD scholarship for A. B. W. Boldt. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Molecular identification of falciparum malaria and human tuberculosis co-infections in mummies from the Fayum depression (Lower Egypt).

Due to the presence of the lake Quarun and to the particular nature of its irrigation system, it has been speculated that the Fayum, a large depression 80 kilometers south-west of modern Cairo, was exposed to the hazards of malaria in historic times. Similarly, it has been speculated that, in the same area, also human tuberculosis might have been far more widespread in the antiquity than in its recent past. If these hypotheses were confirmed, it would imply that frequent cases of co-infection between the two pathogens might have occurred in ancient populations. To substantiate those speculations, molecular analyses were carried out on sixteen mummified heads recovered from the necropolis of Abusir el Meleq (Fayum) dating from the 3(rd) Intermediate Period (1064-656 BC) to the Roman Period (30 BC-300 AD). Soft tissue biopsies were used for DNA extractions and PCR amplifications using well-suited protocols. A partial 196-bp fragment of Plasmodium falciparum apical membrane antigen 1 gene and a 123-bp fragment of the Mycobacterium tuberculosis complex insertion sequence IS6110 were amplified and sequenced in six and five of the sixteen specimens, respectively. A 100% concordance rates between our sequences and those of P. falciparum and M. tuberculosis complex ones were obtained. Lastly, concomitant PCR amplification of P. falciparum and M. tuberculosis complex DNA specific fragments was obtained in four mummies, three of which are (14)C dated to the Late and Graeco-Roman Periods. Our data confirm that the hydrography of Fayum was extremely conducive to the spread of malaria. They also support the notion that the agricultural boom and dense crowding occurred in this region, especially under the Ptolemies, highly increased the probability for the manifestation and spread of tuberculosis. Here we extend back-wards to ca. 800 BC new evidence for malaria tropica and human tuberculosis co-occurrence in ancient Lower Egypt.

Association of L-ficolin levels and FCN2 genotypes with chronic Chagas disease.

BACKGROUND: L-ficolin (encoded by FCN2) binds to acetylated sugar moieties of many pathogens, including Trypanosoma cruzi, promoting their phagocytosis and lysis by the complement system. METHODS: We investigated L-ficolin levels in 160 T. cruzi infected patients with chronic Chagas disease and 71 healthy individuals, and FCN2 polymorphisms (-986 G>A, -602 G>A, and -4 A>G in the promoter and A258S in exon 8) in 243 patients, being 88 indeterminate (asymptomatic), 96 with cardiac, 23 with digestive and 33 with cardiodigestive manifestations (two were unspecified) and 305 controls (135 for A258S). RESULTS: Patients presented lower L-ficolin plasma levels than controls (p<0.0001). Among the different groups of cardiac commitment, individuals with moderate forms had higher L-ficolin levels than the severe forms (P = 0.039). Lower L-ficolin levels were found associated with the 258S variant in the patients (P = 0.034). We found less -4A/G heterozygotes in the cardiac patients, than in the controls (OR = 0.56 [95% CI = 0.33-0.94], P = 0.034). Heterozygote -4A/G genotypes with the 258S variant and 258SS homozygotes were nevertheless more frequent among cardiodigestive patients than in controls (OR = 14.1 [95% CI = 3.5-56.8], P = 0.0001) and in indeterminate patients (OR = 3.2 [95% CI = 1.1-9.4], P = 0.037). We also found an association of the allelic frequency of the 258S variant with cardiodigestive Chagas disease compared to controls (OR = 2.24 [95% CI = 1.1-4.5], P = 0.037). Thus, decreased patient levels of L-ficolin reflect not only protein consumption due to the disease process, but also the higher frequency of the 258S variant in patients with cardiodigestive symptoms. CONCLUSION: The very first study on Brazilian cohort associates both L-ficolin plasma levels and FCN2 variants to Chagas disease and subsequent disease progression. The prognostic value of L-ficolin levels and the FCN2*A258S polymorphism should be further evaluated in other settings.

Plasmodium vivax malaria in Duffy-negative individuals from Ethiopia.

BACKGROUND: This study was conducted to estimate the prevalence of Plasmodium vivax and polymorphisms in the Duffy antigen receptor for chemokines (DARC) gene in patients with suspected malaria from eastern (Harar) and southwestern (Jimma) Ethiopia. METHODS: Plasmodium presence and species was assessed by microscopy in 1304 and 627 febrile patients in Harar and Jimma, respectively, during October-November 2009. All microscopy-positive samples were confirmed by PCR. DARC gene polymorphisms were identified by DNA sequencing. RESULTS: Plasmodium vivax was the dominant species in Harar (74/98, 76%) and P. falciparum was more common in Jimma (70/107, 65%). We found 17/98 (17%) and 24/107 (22%) homozygous Duffy-negative patients in Harar and Jimma, respectively. Unexpectedly, three Duffy-negative patients from Harar had P. vivax malaria. CONCLUSION: This study documents the emergence of P. vivax malaria in Duffy-negative individuals in Ethiopia. The Duffy-negative blood group does not appear to provide absolute protection against P. vivax infection in this region.

Expression of interleukin-6 family receptors in NK92 cells is regulated by cytokines and not through direct interaction with Plasmodium falciparum-infected erythrocytes.

A balanced proinflammatory cytokine response to Plasmodium ssp. infection is crucial to control the disease outcome. To elucidate the effect of cytokines and Plasmodium falciparum-infected erythrocytes on the regulation of interleukin (IL)-6 receptor (IL-6R), ciliary neurotrophic factor alpha (CNTFR-α) and glycoprotein (gp)130 in natural killer (NK) cells in the context of malaria, we assessed their gene expression and surface expression in NK92 cells. P. falciparum alone did not alter gene expression of the investigated receptors in NK92 cells. Analysis revealed a low effect of IL-6 on IL-6R surface expression in NK92 cells. However, at transcriptional level, a downregulation of IL-6R was observed following IL-6 stimulation. Thus, IL-6 might act within a negative feedback loop to terminate signal transduction by downregulating IL-6R expression. Additionally, we observed that IL-6R and CNTFR-α surface expression were regulated by a combination of IL-2, 12, and 18, and gp130 was influenced by interferon-α. Our results show that the IL-6 family receptors in NK92 cells are not directly influenced by P. falciparum. However, cytokines usually derived from accessory cells during malaria episodes may regulate IL-6 receptor signaling pathways. This finding encourages future studies in a more physiological context and with primary cells isolated from humans with and without malaria.

Susceptibility to leprosy is associated with M-ficolin polymorphisms.

PURPOSE: Mycobacterium leprae exploits complement activation and opsonophagocytosis to infect phagocytes. M-ficolin is encoded by the FCN1 gene and initiates the lectin pathway on monocyte surfaces. We investigated FCN1 promoter polymorphisms that could be responsible for the high interindividual variability of M-ficolin levels and for modulating leprosy susceptibility. METHODS: We genotyped rs2989727 (-1981 G > A), rs28909068 (-791 G > A), rs10120023 (-542 G > A), rs17039495 (-399 G > A), rs28909976 (-271IndelT), rs10117466 (-144C > A) and rs10858293 (+33 T > G) in 400 controls and 315 leprosy patients from Southern Brazil, and in 296 Danish healthy individuals with known M-ficolin levels. RESULTS: Ten haplotypes were identified with sequence-specific PCR and/or haplotype-specific sequencing. We found evidence for a protective codominant additive effect of FCN1*-542A-144C with leprosy in Euro-Brazilians (P=0.003, PBf =0.021, OR=0.243 [CI95% =0.083-0.71]), which was independent of age, ethnic group and gender effects (P=0.029). There was a trend for a positive association of the -399A variant in Afro-Brazilians (P=0.022, PBf =0.154, OR=4.151 [CI95% =1.115-15.454], as well as for a negative association of the FCN1*3A haplotype with lepromatous leprosy, compared with less severe forms of the disease (P=0.016, PBf =0.112, OR=0.324 [CI95% =0.123-0.858]). Danish individuals with this haplotype presented M-ficolin levels higher than the population average of circa 1,000 ng/ml, and -542A-144C, which is able to modify the recognition of transcription factors in silico, occurred in individuals with levels under the 25 percentil (P=0.031). CONCLUSIONS: Our data provide the first evidence that FCN1 polymorphisms are associated with leprosy. M-ficolin may represent a novel key to understand the immunopathogenesis of M. leprae infection.

In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population.

BACKGROUND: The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism. METHODS: We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections. RESULTS: We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function. CONCLUSIONS: The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised.

Ficolin-2 levels and FCN2 genetic polymorphisms as a susceptibility factor in schistosomiasis.

BACKGROUND: Human ficolin-2 (L-ficolins) encoded by the FCN2 gene are pattern-recognition proteins involved in innate immunity and are associated with several infectious diseases. METHODS: A Nigerian cohort of 168 Schistosoma haematobium-infected individuals and 192 healthy controls were examined for functional single-nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) using real-time polymerase chain reaction. RESULTS: The FCN2 -986A and -4G alleles were significantly associated with the occurrence of schistosomiasis (P = .0004 for -986G>A; P = .0001 for -4A>G). The heterozygous genotypes (P = .0006 for -986G>A; P = .0002 for -4A>G) were observed to be a risk factor for susceptibility to schistosomiasis, whereas the homozygous genotypes of major alleles (P = .0002 for -986G>A; P = .0001 for -4A>G) were observed to shield against schistosomiasis. The haplotype AGGG (P = .0002) was observed to be a risk factor for susceptibility to schistosomiasis compared with controls, and the haplotype GGAG (P = .04) was observed to confer protection compared with patients. Ficolin-2 serum level was significantly higher in controls (P < .005) and in controls with GGAG haplotypes (P < .0001). CONCLUSIONS: Our findings demonstrate that FCN2 promoter variants (-986G>A and -4A>G) influence ficolin-2 serum levels and susceptibility to schistosomiasis.

Reliable and rapid characterization of functional FCN2 gene variants reveals diverse geographical patterns.

BACKGROUND: Ficolin-2 coded by FCN2 gene is a soluble serum protein and an innate immune recognition element of the complement system. FCN2 gene polymorphisms reveal distinct geographical patterns and are documented to alter serum ficolin levels and modulate disease susceptibility. METHODS: We employed a real-time PCR based on Fluorescence Resonance Energy Transfer (FRET) method to genotype four functional SNPs including -986 G > A (#rs3124952), -602 G > A (#rs3124953), -4A > G (#rs17514136) and +6424 G > T (#rs7851696) in the ficolin-2 (FCN2) gene. We characterized the FCN2 variants in individuals representing Brazilian (n = 176), Nigerian (n = 180), Vietnamese (n = 172) and European Caucasian ethnicity (n = 165). RESULTS: We observed that the genotype distribution of three functional SNP variants (-986 G > A, -602 G > A and -4A > G) differ significantly between the populations investigated (p < 0.0001). The SNP variants were highly linked to each other and revealed significant population patterns. Also the distribution of haplotypes revealed distinct geographical patterns (p < 0.0001). CONCLUSIONS: The observed distribution of the FCN2 functional SNP variants may likely contribute to altered serum ficolin levels and this may depend on the different disease settings in world populations. To conclude, the use of FRET based real-time PCR especially for FCN2 gene will benefit a larger scientific community who extensively depend on rapid, reliable method for FCN2 genotyping.

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70.

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

Genetic evidence of functional ficolin-2 haplotype as susceptibility factor in cutaneous leishmaniasis.

BACKGROUND: Ficolin-2 coded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity. In this study, we analyzed five functional polymorphisms of the FCN2 gene for their possible association with cutaneous leishmaniasis. METHODS: Initially we screened 40 Syrian Arabs for the entire FCN2 gene. We investigated the contribution of FCN2 functional variants in 226 patients with cutaneous leishmaniasis and 286 healthy controls from Syria. Polymorphisms in the promoter regions (-986G/A, -602G/A, -4A/G) of the FCN2 gene were assessed by TaqMan real time PCR, whereas polymorphisms in exon8 (+6359C/T and +6424G/T) were assessed by DNA sequencing. We also measured serum ficolin-2 levels in 70 control Syrian Arabs and correlated the serum concentrations to FCN2 genotypes and haplotypes respectively. RESULTS: Nine new FCN2 variants including two with non synonymous substitutions in exon6 and exon8 were observed. The homozygous genotypes +6424T/T were distributed more in controls and none in patients (P = 0.04). The AGACG haplotype were observed more in patients than in controls (OR = 2.0, 95%CI 1.2-3.4, P = 0.006). The serum ficolin-2 levels were significantly distributed among the reconstructed ficolin-2 haplotypes (P<0.008) and the haplotype AGACG was observed with higher ficolin-2 levels in 70 control individuals. CONCLUSION: Our results demonstrate a significant association of FCN2 AGACG haplotype with cutaneous leishmaniasis in a Syrian Arab population. These first results provide a basis for a future study that could confirm or disprove possible relationships between FCN2 gene polymorphisms with cutaneous leishmaniasis.

Combined promoter haplotypes of the IL10R genes are associated with protection against severe malaria in Gabonese children.

The critical barrier in control of infections remains the failure of the immune system to clear parasites despite antigen recognition. We examined and validated possible association of regulatory immune gene polymorphisms in a cohort of children with mild and severe malaria. We focussed on two precursors of the Interleukin 10 Receptor (IL10R) gene namely the IL10R alpha and IL10R beta that play a fundamental role in initiation of signal transduction. Initial screening across 40 Gabonese adult individuals revealed two promoter variants for the IL10R alpha and three for the IL10R beta precursor, respectively. Validation of these variants for their allelic gene expression by transient transfection assays exhibited an altered expression in rs56356146 and rs7925112 of the IL10R alpha (P < 0.5); rs8178435 and rs999788 in the IL10R beta constructs (P < 0.0001), respectively. We further investigated the functional role of those SNP variants exhibiting altered expression in a cohort of children with mild and severe malaria. We genotyped 145 children with mild and 185 children with severe malaria for IL10R alpha; for IL10R beta, 102 children with mild and 101 children with severe malaria. We found that none of the SNP variants had any significant association neither in children with mild or severe malaria. The haplotype -185/-116 of IL10R alpha (TT) in combination with the haplotype -754/-750 of IL10R beta (AC) contributed towards mild malaria in comparison to severe malaria [TT + AC odds ratio of 0.73 (95% CI 0.56-0.94) P = 0.01]. This study may provide a better understanding on the role of IL10R promoter allelic variants contribution to a protective effect on the development of severe malaria.

Association of CISH -292A/T genetic variant with hepatitis B virus infection.

Cytokine-inducible SRC homology 2 domain protein (CISH) is a suppressor of cytokine signaling that controls interleukin-2 signaling pathway. We investigated the single nucleotide polymorphism (SNP) -292A>T in 473 Vietnamese hepatitis B virus (HBV) carriers and 416 healthy controls. CISH variants at -292A>T were associated to HBV infection (Allelic: OR, 1.22 95% CI, 1-1.49; P = 0.04; Recessive: OR, 1.69 95% CI 1.23-2.54; P = 0.007). A gene dose effect for the risk allele -292T was observed (P = 0.04). The level of interleukin 2 and liver enzymes such as alanine transaminase, aspartate transaminase, total bilirubin, and direct bilirubin were not associated to CISH polymorphism at position -292A>T This study associated the vital role of CISH SNP -292A>T variant to hepatitis B virus infection in a Vietnamese population.

A simplified intravenous artesunate regimen for severe malaria.

BACKGROUND: We compared a conventional empirically derived regimen with a simplified regimen for parenteral artesunate in severe malaria. METHODS: This was a randomized, double-blind, placebo-controlled comparison to assess the noninferiority of a simplified 3-dose regimen (given at 0, 24, and 48 hours) compared with the conventional 5-dose regimen of intravenous artesunate (given at 0, 12, 24, 48, and 72 hours) in African children with Plasmodium falciparum malaria with a prespecified delta of 0.2. The total dose of artesunate in each group was 12 mg/kg. The primary end point was the proportion of children clearing ≥ 99% of their admission parasitemia at 24 hours. Safety data, secondary efficacy end points, and pharmacokinetics were also analyzed. RESULTS: In 171 children (per protocol), 78% of the recipients (95% confidence interval [CI], 69%-87%) in the 3-dose group achieved ≥ 99% parasite clearance 24 hours after the start of treatment, compared with 85% (95% CI, 77%-93%) of those receiving the conventional regimen (treatment difference, -7.2%; 95% CI, -18.9% to 4.4%). Dihydroartemisinin was cleared slightly more slowly in those children receiving the higher 3-dose regimen (7.4 vs 8.8 L/h for a 13-kg child; P 5 .008). CONCLUSIONS: Pharmacodynamic analysis suggests that 3 doses of artesunate were not inferior to 5 doses for the treatment of severe malaria in children. CLINICAL TRIALS REGISTRATION: NCT00522132.

Ficolin-2 levels and FCN2 haplotypes influence hepatitis B infection outcome in Vietnamese patients.

Human Ficolin-2 (L-ficolins) encoded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity and is mainly expressed in the liver. Ficolin-2 serum levels and FCN2 single nucleotide polymorphisms were associated to several infectious diseases. We initially screened the complete FCN2 gene in 48 healthy individuals of Vietnamese ethnicity. We genotyped a Vietnamese cohort comprising of 423 clinically classified hepatitis B virus patients and 303 controls for functional single nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) by real-time PCR and investigated the contribution of FCN2 genotypes and haplotypes to serum Ficolin-2 levels, viral load and liver enzyme levels. Haplotypes differed significantly between patients and controls (P = 0.002) and the haplotype AGGG was found frequently in controls in comparison to patients with hepatitis B virus and hepatocellular carcinoma (P = 0.0002 and P<0.0001) conferring a protective effect. Ficolin-2 levels differed significantly between patients and controls (p<0.0001). Patients with acute hepatitis B had higher serum Ficolin-2 levels compared to other patient groups and controls.The viral load was observed to be significantly distributed among the haplotypes (P = 0.04) and the AAAG haplotype contributed to higher Ficolin-2 levels and to viral load. Four novel single nucleotide polymorphisms in introns (-941G>T, -310G>A, +2363G>A, +4882G>A) and one synonymous mutation in exon 8 (+6485G>T) was observed. Strong linkage was found between the variant -986G>A and -4A>G. The very first study on Vietnamese cohort associates both Ficolin-2 serum levels and FCN2 haplotypes to hepatitis B virus infection and subsequent disease progression.

Limited response of NK92 cells to Plasmodium falciparum-infected erythrocytes.

BACKGROUND: Mechanisms by which anti-malarial immune responses occur are still not fully clear. Natural killer (NK) cells are thought to play a pivotal role in innate responses against Plasmodium falciparum. In this study, the suitability of NK92 cells as models for the NK mechanisms involved in the immune response against malaria was investigated. METHODS: NK92 cells were assessed for several signs of activation and cytotoxicity due to contact to parasites and were as well examined by oligonucleotide microarrays for an insight on the impact P. falciparum-infected erythrocytes have on their transcriptome. To address the parasite side of such interaction, growth inhibition assays were performed including non-NK cells as controls. RESULTS: By performing microarrays with NK92 cells, the impact of parasites on a transcriptional level was observed. The findings show that, although not evidently activated by iRBCs, NK92 cells show transcriptional signs of priming and proliferation. In addition, decreased parasitaemia was observed due to co-incubation with NK92 cells. However, such effect might not be NK-specific since irrelevant cells also affected parasite growth in vitro. CONCLUSIONS: Although NK92 cells are here shown to behave as poor models for the NK immune response against parasites, the results obtained in this study may be of use for future investigations regarding host-parasites interactions in malaria.

Plasmodium falciparum-infected erythrocytes and IL-12/IL-18 induce diverse transcriptomes in human NK cells: IFN-α/ß pathway versus TREM signaling.

The protective immunity of natural killer (NK) cells against malarial infections is thought to be due to early production of type II interferon (IFN) and possibly direct NK cell cytotoxicity. To better understand this mechanism, a microarray analysis was conducted on NK cells from healthy donors PBMCs that were co-cultured with P. falciparum 3D7-infected erythrocytes. A very similar pattern of gene expression was observed among all donors for each treatment in three replicas. Parasites particularly modulated genes involved in IFN-α/ß signaling as well as molecules involved in the activation of interferon regulatory factors, pathways known to play a role in the antimicrobial immune response. This pattern of transcription was entirely different from that shown by NK cells treated with IL-12 and IL-18, in which IFN-γ- and TREM-1-related genes were over-expressed. These results suggest that P. falciparum parasites and the cytokines IL-12 and IL-18 have diverse imprints on the transcriptome of human primary NK cells. IFN-α-related genes are the prominent molecules induced by parasites on NK cells and arise as candidate biomarkers that merit to be further investigated as potential new tools in malaria control.

High prevalence of dhfr triple mutant and correlation with high rates of sulphadoxine-pyrimethamine treatment failures in vivo in Gabonese children.

BACKGROUND: Drug resistance contributes to the global malaria burden. Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) polymorphisms confer resistance to sulphadoxine-pyrimethamine (SP). METHODS: The study assessed the frequency of SP resistance-conferring polymorphisms in Plasmodium falciparum-positive samples from two clinical studies in Lambaréné. Their role on treatment responses and transmission potential was studied in an efficacy open-label clinical trial with a 28-day follow-up in 29 children under five with uncomplicated malaria. RESULTS: SP was well tolerated by all subjects in vivo. Three subjects were excluded from per-protocol analysis. PCR-corrected, 12/26 (46%) achieved an adequate clinical and parasitological response, 13/26 (50%) were late parasitological failures, while 1/26 (4%) had an early treatment failure, resulting in early trial discontinuation. Of 106 isolates, 98 (92%) carried the triple mutant dhfr haplotype. Three point mutations were found in dhps in a variety of haplotypic configurations. The 437G + 540E double mutant allele was found for the first time in Gabon. CONCLUSIONS: There is a high prevalence of dhfr triple mutant with some dhps point mutations in Gabon, in line with treatment failures observed, and molecular markers of SP resistance should be closely monitored.

Novel and functional regulatory SNPs in the promoter region of FOXP3 gene in a Gabonese population.

Parasites exert a selection pressure on their hosts and are accountable for driving diversity within gene families and immune gene polymorphisms in a host population. The overwhelming response of regulatory T cells during infectious challenges directs the host immune system to lose the ability to mount parasite specific T cell responses. The underlying idea of this study is that regulatory single nucleotide polymorphism (SNPs) can cause significant changes in gene expression in functional immune genes. We identified and investigated regulatory SNPs in the promoter region of the FOXP3 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. We identified two novel and one promoter variants in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. Two promoter variants, -794 (C/G) and the other variant -734/-540 (C/T) revealed a significant higher expression of the reporter gene at basal level in comparison to the major allele. The identification of SNPs that modify function in the promoter regions could provide a basis for studying parasite susceptibility in association studies.

Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population.

Lymphatic filariasis (LF) is a parasitic disease caused by threadlike worms of the Brugia and Wuchereria species that live in the human lymphatic system. Regulatory T cells (Tregs) may play a key role in the pathogenesis of LF, and cytotoxic T-lymphocyte antigen-4 (CTLA4) expressed by Tregs is a potential candidate gene because it modulates T-cell activation. A case-control study was performed to establish a potential association of 5 CTLA4 gene promoter single nucleotide polymorphisms (SNPs; rs733618, rs11571316, rs5742909, rs231775, and rs16840252) with the occurrence of LF in an East Malaysian population (320 LF-infected individuals and 150 healthy controls). Polymorphisms were evaluated using TaqMan real-time polymerase chain reaction followed by direct sequencing. LF carriers of the rs733618 AG genotypes (p = 0.02) and those with combined minor allele G carriers (AG + GG; p = 0.01) exhibited a significantly decreased risk for LF. Among the asymptomatic amicrofilaremic cases, positive associations were reported for all genotypes and variants of rs733618 with odds ratios (ORs) ranging from 0.27 to 0.45. In the asymptomatic microfilaremic cases, marker rs231775 exhibited a significant decreased risk, with ORs ranging from 0.50 to 0.57. The study has identified SNPs in the CTLA4 promoter gene that may be functionally linked with susceptibility to LF.